Liquid interferon-beta formulations

ABSTRACT

The present invention relates to liquid formulations of human interferon-β. The formulations are characterized in that they have a buffer with a pH in the weakly acidic to neutral range of between 5 and 8, preferably between over 5.5 and 8, and that they exhibit high stability of the interferon-β in solution while retaining the molecular integrity.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/508,510, filed 26May 2000, now U.S. Pat. No. 6,923,956, which was a §371 ofPCT/EP98/06065, filed on Sep. 23, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to liquid formulations of humaninterferon-β. The formulations are characterized in that they have a pHin the weakly acidic to neutral range between 5 and 8 and that theinterferon-β is highly stable in solution while retaining the molecularintegrity.

2. Description of Related Art

Naturally occurring interferons are species-specific proteins, in somecases glycoproteins, which are produced by various cells of the bodyafter induction with viruses, double-stranded RNA, otherpoly-nucleotides and antigens. Interferons exhibit a large number ofbiological activities such as, for example, antiviral, antiproliferativeand immunomodulatory properties. At least 3 different types of humaninterferons have been identified; they are produced by leucocytes,lymphocytes, fibroblasts and cells of the immune system and termed α-,β-and γ-interferons. Individual types of interferons can furthermore bedivided into a large number of subtypes.

Native, human interferon-β can be prepared commercially bysuperinduction of human fibroblast cell cultures with poly-IC followedby isolation and purification of the interferon-β by chromatographic andelectrophoretic techniques. Proteins or polypeptides which exhibitproperties similar to those of natural interferon-β can also be preparedby recombinant DNA technologies (EP-A-0 028 033; EP-A-0 041 313; EP-A-0070 906; EP-A-0 287 075; Chernajovsky et al. (1984) DNA 3, 297-308;McCormick et al. (1984) Mol. Cell. Biol. 4. 166-172). Recombinant humaninterferon-β can be produced both in eukaryotic cells (for example CHOcells) and by prokaryotic cells (for example E. coli). The interferonsin question are termed interferon-β-1a and interferon-β-1b respectively.In contrast to interferon-β-1b, interferon-β-1a is glycosylated (Goodkin(1994) Lancet 344, 1057-1060).

A prerequisite for the therapeutic use of interferon-β is that it ispharmaceutically formulated so that the protein is storage-stable over aprolonged period while retaining the molecular integrity. Interferon-βis unstable and subject to various degradation reactions. These include,in particular, the cleavage of peptide bonds, deamidation, oxidation ofthe methionin to methionin sulphide, disulphide exchange, and changes inthe sugar side chain which even include deglycosylation.

Owing to the therapeutical benefit of interferons, a series offormulations have been developed in recent years; however, all of themexhibit certain disadvantages. U.S. Pat. No. 4,647,454 (Inter-Yeda Ltd.)describes a formulation of fibroblast interferon-β which can bestabilized by addition of polyvinylpyrrolidone (PVP) in the highlyacidic range (pH 3.5). Other preferred auxiliaries are mannitol, humanserum albumin and acetate buffers. The formulation is freeze-dried andstored at 4° C.

The Japanese Patent Specification 59 181 224 (Sumitomo Chemical Co.)describes an aqueous solution of interferons in which polar amino acidssuch as arginine, asparagine, glutamic acid, glutamine, histidine,lysine, serine and threonine and their sodium salts together with humanserum albumin are employed for stabilizing the interferons.

The international Patent Application WO 95/31213 (Applied ResearchSystems ARS Holding) describes a liquid formulation for interferon-βwhich is stabilized by addition of a polyol, preferably mannitol, and anon-reducing sugar or an amino acid. The formulation furthermorecomprises a buffer (acetate buffer pH 3.0 to 4.0) and human serumalbumin. While formulas with a pH of between 5 and 6 showed an immediateloss in biological activity, the formulas preferred in the patentspecification are sufficiently stable at pH values of 3.0 and 4.0.Moreover, the statement regarding stability only refers to thebiological activity of the formulation, but not to the molecularintegrity of the active ingredient.

The European Patent Application EP 0 215 658 (Cetus Corp.) describes aformulation for recombinant interferon-β in which the bioactive compoundis dissolved in an aqueous medium at a pH of between 2 and 4 withaddition of stabilizers such as human serum albumin or human plasmaprotein fractions and, if appropriate, dextrose. A further patentapplication of Cetus Corp. (WO 89/05 158) describes a formulation forinterferon-β where either glycerin or polyethylene glycopolymers with amean molecular weight of between 190 to [sic] 1600 daltons are employedas stabilizers at a pH of between 2 and 4. Suitable buffer componentswhich are mentioned are glycine, phosphoric acid and citric acid.

The European Patent Application EP 0 217 645 (Cetus Corp.) describespharmaceutical preparations with IL-2 or interferon-β which aredissolved in an excipient medium at pH 7 to 8 and stabilized withaddition of sodium laurate as surfactant. In addition, SDS is alsorequired as further ionic surfactant in order to stabilize thesepreparations.

The European Patent EP 0 270 799 (Cetus Oncology Corp.) describes aformulation for unglycosylated recombinant interferon-β in an inertwater-based excipient medium which comprises non-ionic polymericdetergents as stabilizer.

The European Patent Application EP 0 529 300 (Rentschler BiotechnologieGmbH) describes liquid interferon-β formulations which comprise aconcentration of 30 or 70 MU/ml recombinant IFN-β, sodium chloride andimidazole buffer or sodium phosphate buffer and have a pH of 7.5(Example 3). These formulations are stable with regard to theirbiological activity for 4 weeks at a storage temperature of 25° C.However, the disadvantage of these compositions is that theconcentration of interferon-β used (≧30 MU/ml) is too high for practicalapplications. Moreover, there is no mention in EP-A-0 529 300 of areduction in the stability of liquid interferon-β formulations byaddition of human serum albumin. In contrast, the addition of humanserum albumin is stated as being preferred.

In addition to formulations for interferon-β, there are also describedpharmaceutical dosage forms with interferon-α. The European PatentSpecification 0 082 481 (Schering Corp.) discloses an aqueousformulation intended for freeze-drying which comprises human serumalbumin, in addition to a phosphate buffer and glycine. Alanine ismentioned as further optional constituent. After reconstitution, the pHof the solution is between 7.0 and 7.4. A further patent application ofSchering Corp. (WO 96/11018) discloses stable aqueous solutions ininterferon-α which comprise chelating agents (NaEDTA or citric acid), asurfactant (Polysorbat 80), an isotonizing agent (sodium chloride) andsuitable preservatives such as methylparaben, propylparaben, m-cresol orphenol, at a pH of between 4.5 and 7.1. With regard to the biologicalactivity (standard method of inhibiting the cytopathic effect (CPE) of avirus as described by W. P. Protzman in J. Clinical Microbiology, 1985,22, pp. 596-599), the aqueous formulations disclosed prove to be stablefor 6 months at 25° C. (biological activity >90% of the initialactivity). However, a determination of the protein content by HPLCcarried out in parallel already shows losses in content of between 20.2(Table 3) or 32.5% (Table 4) after 6 months at 25° C.

EP-A-0 736 303 (Hoffmann-LaRoche AG) discloses aqueous interferon-αcompositions which, in addition to an interferon-α, comprise a non-ionicdetergent, a buffer for setting the pH range between 4.5 and 5.5, benzylalcohol and, if appropriate, isotonizing agents. A determination by HPLCidentifies a residual content of 84.5% after storage for three months at25° C. and a starting concentration of 18 MU interferon-α2a, while thisvalue drops to 62.8% when the stabilizer benzyl alcohol is omitted.

EP-A-0 641 567 (Ciba Geigy AG) describes pharmaceutical compositionswhich comprise hybrid interferon-α and, as stabilizer, a buffer with apH of between 3.0 and 5.0.

U.S. Pat. No. 5,358,708 (Schering Corp.) describes aqueous formulationsof interferon-α which comprise methionine, histidine or mixtures ofthese as stabilizer. After storage of an interferon-α solution at 40° C.for two weeks, it is found that the active ingredient content hasdecreased by 20%.

The abovementioned formulations for interferons have shortcomings fromthe present-day view since, for example, an addition of human serumalbumin for stabilizing proteins should be dispensed with, owing to thehigher demands for safety from virus contamination by blood donors.Moreover, a number of the above-described formulations require theaddition of amino acids and/or freeze-drying. However, freeze-driedproducts are complicated to produce and, accordingly, expensive andrequire an additional pass owing to the necessity of reconstitution, andthis additional pass is frequently very difficult to perform, inparticular for patients with a limited power of movement. A series offormulas have unphysiological pH values of below 5.0. While such valuesare not entirely unusual (see also S. Sweetana and N. J. Aders, Journalof Pharmaceutical Sciences and Technology, 1996, 50: 330-342), painfulirritation must be expected in the case of intramuscular or subcutaneousapplication. While according to Sweetana and Akers the use ofsurfactants such as Polysorbat 80 is admissible, a series of sideeffects have been described, in particular in new-born and olderchildren, which make the use of such auxiliaries questionable. A reviewof the toxicity of surfactants can be found in Attwood and Florence(Surfactant Systems, their Chemistry, Pharmacy and Biology, Chapman andHall; London, 1983). The pharmacology of Polysorbat 80 is reviewed by R.K. Varma et al. (Arzneim.-Forsch./Drug Res. 35, 1985, 804-808).

On the basis of the abovementioned disadvantages, an optimal formulationfor interferon-β should combine the following properties:

-   -   retaining the biological activity over the storage period,    -   retaining the molecular integrity of the active ingredient        molecule over the storage period,    -   liquid formulation, no expensive freeze-drying and no additional        reconstitution,    -   no risky auxiliaries such as human serum albumin or surfactants        (detergents),    -   pH in the neutral to weakly acidic range.

BRIEF SUMMARY OF INVENTION

A first aspect of the present invention is therefore a liquidpharmaceutical formulation which comprises human interferon-β as activeingredient in a concentration of up to 25 MU/ml and a buffer for settinga pH of between 5 and 8, preferably between over 5.5 and 8, is free fromhuman serum albumin and shows a long-term stability of the biologicalactivity (in vitro) of at least 80% of the initial activity afterstorage for 3 months at 25° C.

A further aspect of the invention is a liquid pharmaceutical formulationwhich comprises human interferon-β as active ingredient and a buffer forsetting a pH of between 6 and 7.2, is free from human serum albumin andshows a long-term stability of the biological activity (in vitro) of atleast 80% of the initial activity after storage for 3 months at 25° C.

Yet a further aspect of the invention is a liquid pharmaceuticalformulation which comprises human IFN-β as active ingredient, a bufferfor setting a pH of between 5 and 8, preferably between over 5.5 and 8,and one or more amino acids and shows a long-term stability of thebiological activity (in vitro) of at least 80% of the initial activityafter storage for 3 months at 25° C.

All requirements are met by the invention, which is described in greaterdetail in the section which follows.

DETAILED DESCRIPTION OF INVENTION

Surprisingly, a composition of a formula has been found which ensuresthe molecular integrity of interferon-β in liquid form over a prolongedperiod in a physiological pH range of between 5 and 8, preferablybetween over 5.5 and 8, without having to resort to the auxiliaries ofthe prior art, which are known as being disadvantageous.

The long-term stability of liquid pharmaceutical formulations wasmeasured at 25° C. The temperature of 25° C. was chosen, on the onehand, to cause accelerated degradation reactions, but, on the otherhand, to avoid artefacts caused by unduly high temperatures. Suitableanalytical methods for determining the stability of interferon-β can befound in the reviews by J. Geigert (J. Parent. Sci. Technol. 43 (1989),220-224) or M. C. Manning, K. Patel and R. T. Borchardt (Pharm. Res. 6(1989), 903-918).

The biological activity after the storage period chosen in each case wasmeasured by the standard method of inhibiting the cytopathic effect of avirus. A detailed description of the test method used can be found inStewart, W. E. II (1981): The Interferon System (Second, enlargedEdition), Springer-Verlag: Vienna, New York; Grossberg, S. E. et al.(1984), Assay of Interferons. In: Came, P. E., Carter W. A (eds)Interferons and their Applications, Springer-Verlag: Berlin, Heidelberg,New York, Tokyo, pp. 23-43. After storage for three months at 25° C., aformulation according to the invention exhibits a biological activity ofat least 80%, preferably of at least 85%, and especially preferably ofat least 90%, of the initial activity.

After storage for six months at 25° C., a formulation according to theinvention preferably has a biological activity of at least 80%, andpreferably of at least 85%, of the initial activity.

Even when stored at higher temperatures, for example 37° C., theformulations according to the invention exhibit a surprisingly highlong-term stability of the biological activity. For example afterstorage for one month at 37° C., a biological activity of at least 70%,and preferably of at least 80%, of the initial activity is found.

The liquid pharmaceutical formulations according to the invention arepreferably free from human serum albumin and especially preferably—apartfrom the active ingredient—free from human or animal polypeptides, inparticular serum proteins. It is furthermore preferred for the liquidpharmaceutical formulation according to the invention to be free fromsurfactants, in particular free from ionic detergents and/or non-ionicsurfactants.

The formulations according to the invention comprise, as activeingredient, an interferon-β, that is to say a polypeptide which exhibitsbiological and/or immuno-logical properties of natural humaninterferon-β and which may be a naturally occurring or recombinantinterferon-β. The formulation preferably comprises a glycosylatedinterferon-β, especially preferably a recombinant interferon-β from CHOcells. Interferon-β species which are most preferably used are thosewhich can be obtained from the cell line BIC 8622 (ECACC 87 04 03 01)and which are described, for example, in EP-B-0 287 075 and EP-A-0 529300.

Preferably, the active ingredient is present in the formulationsaccording to the invention in a concentration of up to 25 MU/ml.However, a dosage in the range of 1 to 25 MU/ml is preferred, in therange of 3 to 20 MU/ml especially preferred and in the range of 3 to 10MU/ml most preferred. These dosage ranges allow an immediate use withoutfurther dilution in conjunction with a particularly good stability at anelevated temperature.

A further preferred feature of the liquid pharmaceutical formulationaccording to the invention is that it exhibits a chemical integrityafter storage for 3 months, and preferably 6 months, at 25° C., i.e.that it is stable to peptide cleavage, oxidation and deglycosylation.The chemical integrity is measured by peptide mapping, Western blot andglycosylation analysis. Chemically stable for the purposes of thepresent invention are compositions in which the interferon-β afterformulation retains at least 85%, preferably at least 90%, of thechemical integrity at the storage conditions chosen.

A further preferred feature of the liquid pharmaceutical formulationsaccording to the invention is a physical integrity after storage for 3months, and preferably 6 months, at 25° C. The physical integrity is inthis case measured by measuring the transmission at 420 nm and byvisually observing the solutions. Physically stable are those solutionswhose transmission is over 90%, preferably over 93%, at the storageconditions chosen, and where no turbidity can be determined upon visualobservation.

The present invention surprisingly allows liquid formulations ofinterferon-β to be provided which are biologically, chemically andphysically stable over a prolonged period and free from undesiredconstituents such as, for example, human serum albumin or surfactants.In addition to the active ingredient, the formulations according to theinvention comprise a buffer which is preferably present in aconcentration of 10 mmol/l to 1 mol/l, especially preferably in aconcentration of 20 mmol/l to 200 mmol/l, for example approximately 50mmol/l to 100 mmol/l, and which serves to maintain the pH of theformulation in the range of 5 to 8, preferably above 5.5 to 8, morepreferably between 6 and 7.4. A pH range between 6 and 7.2 is especiallypreferred, and a pH range between 6.2 and 6.8 most preferred, since aparticularly high stability while retaining the molecular integrity isachieved here. The buffer is selected from amongst pharmaceuticallyacceptable buffers, for example borate, succinate, L-malate, TRIS,salicylate, glycyl-glycine, triethanolamine, isocitrate, maleate,phosphate, citrate and acetate buffer, or mixtures of these. Phosphate,citrate and acetate buffer or mixtures of these are preferably used,especially preferably phosphate/citrate buffers.

In addition to the active ingredient and the buffer, the formulationaccording to the invention can comprise other physiologically acceptableauxiliaries, for example auxiliaries for adapting tonicity to thetonicity of blood or tissue, for example non-reducing sugars, sugaralcohols such as mannitol, sorbitol, xylitol or glycerin. Moreover, oneor more amino acids such as, for example, alanine, arginine, glycine,histidine or/and methionine, may be added to the formulation accordingto the invention to further increase the chemical stability. Methionineis preferred in this context. The methionine concentration is preferablyin the range of 0.1 to 4 mmol/l. A concentration of 2 mmol/l isespecially preferred. However, the formulation does not comprise anyacidic amino acids, arginine or glycine in amounts of between 0.3 and 5%by weight. Moreover, the composition may comprise thickeners forincreasing the viscosity, for example for ophthalmological purposes.Examples of suitable thickeners are ophthalmologically suitablepolymers, for example Carbopol, methylcellulose, carboxymethylcelluloseetc.

Moreover, the composition according to the invention may also comprisepreservatives. For ophthalmological purposes, for example, thiomersalatemay be employed in an amount of 0.001 to 0.004% (weight/volume).

The invention furthermore relates to pharmaceutical preparations whichcomprise a liquid interferon-β-comprising formulation as describedabove. These pharmaceutical preparations are particularly suitable fororal, parenteral or ophthalmological application. The formulationspreferably exist in unit doses of 1 to 25 MU IFN-β. The inventionfurthermore relates to a process for the preparation of suchpharmaceutical preparation, in which a formulation according to theinvention and, if appropriate, other pharmaceutical formulationauxiliaries which are necessary are prepared and formulated as asuitable dosage form.

The formulation according to the invention can be stored in suitable,washed and sterilized glass vials (hydrolytic class 1) withpharmaceutically acceptable rubber stoppers.

Moreover, formulations according to the invention can also be packagedaseptically in ready-to-use syringes or else in carpules for use inself-injection systems, and employed thus. While this is not preferred,the aqueous solutions may be freeze-dried by addition of otherauxiliaries known to the skilled worker, and, after reconstitution, areavailable in liquid form.

Using suitable preservatives, it is possible to prepare liquid multidoseforms and eye-drop solutions and solutions for dropwise oralapplication.

The auxiliaries additionally required for preparing the relevant dosageforms are known to the skilled worker.

Finally, the invention relates to a process for improving the shelf lifeof a liquid formulation which comprises human interferon-β as activeingredient and a buffer for setting a pH of 5 to 8, preferably of above5.5 to 8, characterized in that a formulation without human serumalbumin or/and with one or more amino acids is used. The improvement inshelf life encompasses improved long-term stability of the biologicalactivity (in vitro), of the chemical integrity or/and of the physicalintegrity as indicated hereinabove.

The invention is furthermore illustrated by the examples which follow.

EXAMPLES

An interferon-β obtained from CHO cells was used in all the examples.

1. Long-term Stability of Liquid Interferon-β Formulations at 25° C.

The following formulations were tested:

-   Formulation 1: 50 mmol/l sodium citrate pH 5.0-   Formulation 2: 50 mmol/l sodium citrate, 50 mmol/l sodium phosphate    pH 7.0, 15 mg/ml human serum albumin, 2 mmol/l methionine, 50 mg/ml    glycerin-   Formulation 3: 50 mmol/l sodium citrate, 50 mmol/l sodium phosphate    pH 7.0, 50 mg/ml glycerin, 2 mmol/l methionine-   Formulation 4: 50 mmol/l sodium citrate, 50 mmol/l sodium phosphate    pH 7.0, 2 mmol/l methionine-   Formulation 5: 50 mmol/l sodium citrate, 50 mmol/l sodium phosphate    pH 7.0-   Formulation 17: 70 mmol/l sodium citrate, 50 mmol/l sodium    phosphate, 2 mmol/l methionine, pH 6.5

The formulations were diluted to a content of approx. 10 to 15 MU/ml(that is to say 10 to 15×10⁶ IU/ml.

With the exception of formulation 17 (see below), the formulations werestored at 25° C. for the period indicated in hydrolytic class 1 glassvials (DIN 2R vials) which were sealed with commercially availablechlorobuthyl rubber stoppers. The biological activity (in vitro) wasdetermined as described by Stewart, W. E. II (1981): The InterferonSystem (Second, enlarged edition) Springer-Verlag: Vienna, New York;Grossberg, S. E. et al. (1984) Assay of Interferons. In: Came, P. E.,Carter W. A. (eds.) Interferons and their Applications, Springer-Verlag:Berlin, Heidelberg, New York, Tokyo, pp. 23-43.

The results are shown in Tables 1 to 5. “% (ref.)” indicates thebiological activity based on the biological activity of a referencesample which had been stored at −20° C. for the period indicated. “%(0mo)” is the percentage of biological activity based on the initialvalue at 0 months.

TABLE 1 (Formulation 1): Active ingredient content MU/ml Recovery (25°C.) Months −20° C. 25° C. % (ref.) % (0 mo.) 0 11.0 11.0 100 100 1 10.09.8 98 89 2 9.7 11.0 113 100 3 10.0 10.6 106 96 4 10.3 9.5 92 86 5 9.59.7 102 88 6 10.5 10.2 97 93

TABLE 2 (Formulation 2): Active ingredient content MU/ml Recovery (25°C.) Months −20° C. 25° C. % (ref.) % (0 mo.) 0 13.9 13.9 100 100 1 14.011.9 85 86 2 13.0 11.6 89 83 3 13.1 9.6 73 69 4 12.5 8.8 70 63 5 11.08.2 75 59 6 13.3 8.4 63 60

TABLE 3 (Formulation 3): Active ingredient content MU/ml Recovery (25°C.) Months −20° C. 25° C. % (ref.) % (0 mo.) 0 12.5 12.5 100 100 1 9.410.0 106 80 2 8.3 11.5 139 92 3 7.8 11.8 151 94.4 4 6.8 10.3 151 82.4 56.6 11.2 170 89.6 6 7.8 13.4 172 107.2

TABLE 4 (Formulation 4): Active ingredient content MU/ml Recovery (25°C.) Months −20° C. 25° C. % (ref.) % (0 mo.) 0 11.4 11.4 100 100 1 10.510.2 97 89 2 11.9 11.1 93 97 3 10.8 10.0 93 88 4 10.4 9.3 89 82 5 11.68.4 72 74 6 12.4 9.5 77 83

TABLE 5 (Formulation 5): Active ingredient content MU/ml Recovery (25°C.) Months −20° C. 25° C. % (ref.) % (0 mo.) 0 11.3 11.3 100 100 1 11.09.7 88 86 2 11.7 10.1 86 89 3 11.1 10.2 92 90 4 11.3 10.2 90 90 5 12.09.2 77 81 6 11.0 9.7 88 86

It can be seen from the above tables that formulations which do notcontain human serum albumin (Formulations 1, 3, 4, 5) surprisinglyexhibit a better stability than a formulation which comprises humanserum albumin (Formulation 2).

In Formulation 17 (see above), an interferon solution without humanserum albumin was brought to an activity of 6 MU/0.5 ml under asepticconditions. The colourless, clear solution was subsequentlyfilter-sterilized, and 0.5-ml aliquots were filled into pre-sterilizeddisposable syringes and sealed. The ready-to-use syringes were stored at25° C. and examined for clarity, pH and biological activity. Thefollowing results were obtained:

Storage in MU/syringe Recovery (25° C.) months pH Clarity[%] −20° C. 25°C. % (ref.) % (0 mo.) 0 6.5 99.5 6.3 6.3 100 100 3 6.5 99.1 5.6 6.1 108 972. Long-term Stability of Liquid IFN-β Formulations at 37° C.

The following formulations in ready-to-use syringes were tested:

-   Formulation 6: 50 mmol/l sodium citrate, 50 mmol/l sodium phosphate    pH 7.0, 2 mmol/l methionine-   Formulation 7: 50 mmol/l sodium citrate pH 5.0, 18 mg/ml glycerin, 2    mmol/l methionine-   Formulation 8: 50 mmol/l sodium citrate pH 5.0, 18 mg/ml glycerin,    15 mg/ml human serum albumin, 2 mmol/l methionine-   Formulation 9: 50 mmol/l sodium citrate pH 6.0, 18 mg/ml glycerin, 2    mmol/l methionine-   Formulation 10: 50 mmol/l sodium citrate pH 6.5, 18 mg/ml glycerin,    2 mmol/l methionine

The formulations were tested in dosage strengths of 3 MU per 0.5 ml(dosage strength 3), 6 MU per 0.5 ml (dosage strength 6) and 12 MU perml (dosage strength 12).

The results are shown in Table 6 which follows.

TABLE 6 Storage Dosage strength 3 Dosage strength 6 Dosage strength 12in Formulation Formulation Formulation months 6 7 8 9 10 6 7 8 9 10 6 78 9 10 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 171 80 61 74 69 72 85 63 86 84 87 88 71 76 84 2 51 82 33 74 85 61 81 4380 76 69 88 48 77 81 3 44 76 23 63 65 48 64 36 73 69 66 72 35 80 81 4 3351 16 61 61 46 65 26 84 — — 64 24 78 79

The results of Table 6 show that, surprisingly, the formulationsaccording to the invention without human serum albumin exhibit animproved stability at 37° C.

3. Chemical Stability at 25° C.

To examine the chemical stability of liquid formulations of IFN-β, 7batches were formulated and stored at 25° C. After 3 and 6 months, theprotein was characterized by means of Lys-C mapping and completecarbohydrate analysis. The formation of methionine sulphoxide and thedesialylation was checked particularly carefully.

In addition to Formulation 10 (see above), the following formulationswere tested:

-   Formulation 11: 50 mmol/l sodium citrate, 50 mmol/l sodium    phosphate, 2 mmol/l methionine pH 7.0 to 7.2-   Formulation 12: 50 mmol/l sodium citrate, 50 mmol/l sodium phosphate    pH 7.0 to 7.2-   Formulation 13: 50 mmol/l sodium citrate, 18 mg/ml glycerin, 2    mmol/l methionine, pH 5.0 to 5.2-   Formulation 14: 50 mmol/l sodium citrate, 18 mg/ml glycerin, pH 5.0    to 5.2-   Formulation 15: 50 mmol/l sodium citrate, 15 mg/ml human serum    albumin (medical grade), 18 mg/ml glycerin, 2 mmol/l methionine, pH    5.0 to 5.2-   Formulation 16: 50 mmol/l sodium citrate, 15 mg/ml human serum    albumin (medical grade), 18 mg/ml glycerin, pH 5.0 to 5.2    (comparison)

In all batches, the IFN-β content was between 10 and 11 MU/ml.

Testing Procedure

To carry out the analyses, the samples had to be concentrated. Moreover,the human serum albumin had to be removed in the case of batches 15 and16. This is why the batches were passed over an anti-β chromatographycolumn. The initial volume per batch was 32 ml. Batches 13 to 16 wereneutralized prior to anti-β chromatography by addition of 2.1 ml of 0.4mol/l Na₂HPO₄ and 2.1 ml of 0.4 mol/l Na₃PO₄.

For the immunoadsorption of interferon-β on a monoclonal antibodyagainst interferon-β (BO2 sepharose 6B, crosslinked by Celltech), a C10chromatography column (Pharmacia) was packed with 5 ml of BO2 sepharoseand washed 3 times with in each case 5-10 gel volumes of PBS, 0.1 mol/lsodium phosphate pH 2.0 and PBS/1 mol/l KCl at a linear flow rate of 1.0cm/min.

Approximately 32 ml of the interferon/HSA-containing solution wasapplied at a linear flow rate of 0.5 cm/min.

Washing was effected with 10 gel volumes of PBS/1 mol/l KCl with alinear flow rate of 1 cm/min until the OD had dropped to baseline.Elution was done with approximately 1-2 gel volumes of 0.1 mol/l sodiumphosphate pH 2.0 at a linear flow rate of 1 cm/min. Interferon-β isobtained as single peak-in high purity. This eluate is suitable for thesubsequent protein characterization.

Analytical Procedure

1. Lys-C Mapping

Using the Achromobacter (AP) enzyme endoproteinase Lys-C, interferon-βis cleaved under reducing conditions on the C-terminal end of lysin togive 12 peptides.

50 μl of eluate from the anti-β chromatography 12.5-50 μg ofinterferon-β) were placed into an Eppendorf reaction vessel, and 5 μl of2 mol/l TRIS were added. Wako endoproteinase was added in anenzyme/substrate ratio of 1:10 (endoproteinase Lys-C solution in 50mmol/l TRIS/HCl, pH 9.0). The solution was mixed and incubated for 2hours at 30° C. Then, 5 μl of 0.1 mol/l DTT were added to the batch.

The peptides were separated on a reversed-phase column (Vydac C18, 300Å,5 μm, 2.1 mm) on an HPLC system HP 1090 M series with diode arraydetector at 214 mm, for which purpose a gradient of A: 0.1% (v/v) TFAand B: 0.1 (v/v) TFA/70% (v/v) acetonitrile was used. The peptides werenumbered consecutively in the sequence of their retention times and areallocated to the following sequences:

SEQ. ID. No. Peptide Position Sequence 1 AP1 109-115 EDFTRGK 2 AP2100-105 TVLEEK 3 AP3 46-52 QLQQFQK 4 AP4(ox) 116-123 LM(ox)SSLHLK 5 AP4116-123 LMSSLHLK 6 AP6(ox) 35-45 DRM(ox)NFDIPEEIK 7 AP5 124-134RYYGRILHYLK 8 AP6 34-45 DRMNFDIPEEIK 9 AP7 20-33 LLWQLNGRLEYCLK 10AP8(ox)  1-19 M(ox)SYNLLGFLQRSSNFQCQK 11 AP8  1-19 MSYNLLGFLQRSSNFQCQK12 AP9 137-166 EYSHCAWTIVRVEILRNFYFINRLTG YLRN 13 AP10(ox) 53-99EDAALTIYEM(ox)LQNIFAIFRQDS SSTGWNETIVENLLANVYHQINHLK 14 AP10 53-99EDAALTIYEMLQNIFAIFRQDSSSTG WNETIVENLLANVYHQINHLKReferences:

-   Utsumi et al. (1989). Characterization of four different    mammalian-cell-derived recombinant human interferon-β1. Eur. J.    Biochem. 181, 545-553.-   Utsumi et al. (1988): Structural characterization of fibroblast    human interferon-β1. J. Interferon Res. 8, 375-384-   Allen, G. (1981): Laboratory techniques in biochemistry and    molecular biology. Sequencing of proteins and peptides. Elsevier    Verlag.-   Castagnola et al. (1988): HPLC in Protein sequence    determinations. J. Chromatography 440, 213-251.

In the peptides marked (ox), the amino acid methionine is in the form ofmethionine sulphoxide. The quantification is based on determining theproportion of the peak area of the oxidized peptide relative to thetotal area of intact peptide and oxidized peptide. The proportions ofoxidized methionines are very low in fresh interferon-β preparations.However, the proportion increases more or less drastically duringstorage, depending on the storage conditions (buffer, pH, temperatureetc.). This change is undesired since it contributes to the instabilityof the interferon-β molecule or can significantly affect the in-vivoproperties.

The proportion of the oxidized peptides AP4 (ox), AP6(ox), AP8(ox) andAP10(ox) is thus an important criterion for assessing the chemicalintegrity of the interferon-β molecule in a liquid formulation.

2. Carbohydrate Determination

In the first step, the oligosaccharides were separated from thepolypeptide and demineralized.

Approximately 0.7 ml of the eluate of the anti-β chromatography weredialysed for 16-20 hours at room temperature in a dialysis tubing(diameter 6 mm, Sigma No. D-9277) against 500 ml of dialysis buffer(0.05 mol/l sodium phosphate, 0.10 mol/l NaCl, pH 7.25), with gentlestirring. Then, the tubing was cut open at one end and the contentssqueezed into a Eppendorf reaction vessel. After dialysis, the samplevolume was 1 ml.

20 μl of Tween 20 (10% strength) and 15 μl of N-glycosidase F solution(Boehringer Mannheim) were pipetted to the dialysed sample. This mixturewas incubated for 24 hours at 37° C. After the incubation had ended, themixture was centrifuged for 10 minutes at 10,000 rpm, filtered through a0.45 μm filter and subsequently chromatographed and fractioned over adesalting column (HR 10/10 Pharmacia No. 17-0591-01) with an isokraticgradient (eluent A: distilled water) at a flow rate of 1.0 ml/min. Thefree oligosaccharides were detected at 206 nm.

In the second step, the oligosaccharides which had been liberated wereseparated by an ion exchanger as a function of the number of the sialicacid residues.

The oligosaccharides contained in the eluate of the desalted column,approx. 2 ml, were bound to an anion exchanger (Mono Q HR 5/5, PharmaciaNo. 17-0546-01). The asialo forms are in the eluate. With the aid of ashallow NaCl gradient, monosialo, disialo and trisialo forms eluteddistinctly separately one after the other in the sequence indicated.

-   Eluent A: Milli-Q water-   Eluent B: 0.10 mol/l NaCl    Gradient

 0 min 100% A  0% B  5 min 100% A  0% B 25 min  33% A 67% B 26 min 100%A  0% B

-   Flow rate: 0.75 ml/min-   Chromatography time: 26 min (with regeneration 36 min)-   Detection: UV 206 nm

The individual oligosaccharide fractions were detected by means of a UVdetector at 206 nm. The quantitative calculation was done by integratingthe areas of the individual peaks.

The oligosaccharide fractions monosialo, disialo and trisialo weresubsequently passed over a desalting column as described above.

In the third step, the charged oligosaccharides are converted intoneutral oligosaccharides by hydrolytically eliminating the terminalsialic acid residues under acidic pH conditions.

To this end, approx. 15 μl of each oligosaccharide fraction plus 15 μlof Milli Q water were placed into a micro-test tube, and 30 μl of 10mmol/l H₂SO₄ were added. The mixture was then heated for 90 minutes at80° C.

Then, the batch was centrifuged for 1-minute at 5000 rpm and pipettedinto a minivial. The carbohydrates, which are now neutral, are bound atalkaline pH to weak anions and on an anion-exchanger column (CarboPacPA1 (4×250 mm) P/N 35391, Dionex). Elution is done with a gradient of

-   Eluent A: NaOH 0.16 mol/l-   Eluent B: NaOH 0.16 mol/l sodium acetate 0.10 mol/l-   Eluent C: NaOH 0.16 mol/l sodium acetate 0.75 mol/l    Gradient:

  0 min 95% A  5% B  0% C  2.0 min 95% A  5% B  0% C  3.0 min 85% A 15%B  0% C  4.0 min 85% A 15% B  0% C 28.0 min 37% A 63% B  0% C 28.1 min90% A  0% B 10% C 45.0 min 20% A  0% B 80% C 45.1 min 95% A  5% B  0% C50.0 min 95% A  5% B  0% C

-   Flow rate: 1.0 ml/min-   Chromatography time: 50 min-   Detection: PAD

PAD (pulsed amperometric detection) was used to determine theoligosaccharides. The oligosaccharide molecule is electrochemicallyoxidized, and the current thus formed measured. PAD is distinguished bya high sensitivity, so that a detection in the ng range presents nodifficulty. The output signal in the detector (in mV) is directlyproportional to the amount of carbohydrate. Quantification is done byintegrating the peak areas.

Between the deglycosylation and the analysis, the samples were subjectedto intermediate storage at −20° C.

References:

-   Townsend (1988): High-performance anion-exchange chromatography of    oligosaccharides. Analytical Biochemistry 174, 459-470.    Results    1. Lys-C Mapping

The Lys-C mapping of batches 11 to 16 showed no difference to theinitial value with regard to retention time and qualitativedetermination of the peptides.

The determination of the methionine sulphoxide content during liquidstorage revealed the results shown in Tables 7 (3 months' storage) and 8(6 months' storage).

TABLE 7 AP4ox AP6ox AP8ox AP10ox Name content content content content tovalue  <5% 7.6% n.d. n.d. Formulation 11 7.9% 10.5% n.d. n.d.Formulation 12  <5% 11.6% n.d. n.d. Formulation 13  <5% 7.3% n.d. n.d.Formulation 14  <5% 9.4% n.d. n.d. Formulation 15  <5% 8.6% n.d. n.d.Formulation 16  <5% 10.8% n.d. n.d. (n.d. = not detectable)

TABLE 8 AP4ox AP6ox AP8ox AP10ox Name content content content content tovalue   <5% 7.6% n.d. n.d. Formulation 10  7.6% 8.9% n.d. n.d.Formulation 11  7.7% 9.5% n.d. n.d. Formulation 12 12.0% 13.7% n.d. n.d.Formulation 13  7.4% 8.7% n.d. n.d. Formulation 14 13.7% 15.7% n.d. n.d.Formulation 15  7.4% 7.9% n.d. n.d. Formulation 16 18.0% 17.6% n.d. n.d.

Table 7 reveals that the methionine-containing batches 13 and 15 show alower methionine sulphoxide content upon three months' storage incomparison with methionine-free batches. After storage for six months,the affect of the added methionine in batches 11, 13 and 15 is morepronounced. Only a very small increase in the methionine sulphoxidecontent can be detected in these batches. In the methionine-freebatches, the methionine sulphoxide content increases slightly more, butthe total of all oxidized methionine contents amounts to less than 10%of the total methionine content.

2. Carbohydrate Determination

The results of the carbohydrate determination after storage for three or6 months are shown in Tables 9a, 9b, 10a, 10b, 11a and 11b.

Interferon-β-1a has a carbohydrate structure on its amino acid chainwhich is composed of a defined sequence of monosaccharides. Depending onthe type of branching, these structures are termed biantennary (2 arms),triantennary (3 arms) and tetraantennary (4 arms).

The carbohydrate structure is composed of the monosaccharides mannose,fucose, N-acetylglucosamine, galactose and sialic acid.

In this context, the sialic acid is special in several respects:

-   -   It is the only monosaccharide with a charged group (carboxyl        group).    -   It always occurs at the terminus of the carbohydrate chain.    -   It can be eliminated enzymatically or hydrolytically        considerably more readily than the remaining monosaccharides.    -   While the structure of the neutral carbohydate chain is highly        constant, the sialic acid moiety varies greatly depending, inter        alia, on the cell culture and the purification method of the        interferon.        References:

-   Kagawa et al., J. Biol. Chem. 263 (1988), 17508-17515; EP-A-0 529    300.

The sialostatus (percentage of individual sialo structures) after threemonths' storage (Table 9a) or six months' storage (Table 9b) wasinvestigated. A carbohydrate structure which does not contain a terminalsialic acid is termed asialo. A carbohydrate structure which contains aterminal sialic acid is termed monosialo. A carbohydrate structure whichcontains two terminal sialic acids is termed disialo. A carbohydratestructure which contains three terminal sialic acids is termed trisialo.

Furthermore, the antennarity (percentage of individual branching types)was determined after three months' storage (Table 10a) and after sixmonths' storage (Table 10b). A carbohydrate structure with one branchingand thus two terminal galactoses is termed biantennary. It can have zeroto two terminal sialic acids. A carbohydrate structure with twobranchings and thus three terminal galactoses is termed triantennary. Itcan have zero to three terminal sialic acids.

The degree of sialylation (percentage occupation of terminal galactoseresidues with sialic acid) after three months' storage (Table 11a) andsix months' storage (Table 11b) was also investigated.

It can be seen from the results that storage at pH 5 causes a slight,but reproducible, desialylation. Storage at pH 7 has no effect on thedegree of sialylation.

The afuco content specified in batches 15 and 16 is probably due toforeign proteins from the added human serum albumin, which were notquantitatively removed by anti-β chromatography.

As regards the antennarity, liquid storage has no measureable effect.

TABLE 9a Name Asialo Monoasialo Disialo Trisialo to value <3 13.4 73.412.1 Formulation 11 <3 14.0 74.1 11.9 Formulation 12 <3 12.6 74.9 11.6Formulation 13 <3 16.5 70.4 12.0 Formulation 14 <3 16.6 71.1 11.1Formulation 15 <3 15.8 70.0 13.0 Formulation 16 <3 15.1 72.0 11.9

TABLE 9b Name Asialo Monoasialo Disialo Trisialo to value <3 13.4 73.412.1 Formulation 10 <3 13.9 70.2 15.3 Formulation 11 <3 14.5 73.9 11.6Formulation 12 <3 14.0 72.4 13.6 Formulation 13 <3 18.6 68.9 11.7Formulation 14 <3 19.0 69.4 10.7 Formulation 15 <3 17.0 71.0 11.3Formulation 16 <3 16.1 71.5 12.4

TABLE 10a Triantennary Triantennary + Name Biantennary 1 → 6 1 repeat tovalue 74.4 18.1 3.7 Formulation 11 72.9 18.7 3.7 Formulation 12 76.917.0 2.7 Formulation 13 74.7 18.0 3.1 Formulation 14 75.9 17.3 2.9Formulation 15 76.2 18.0 3.3 (incl. 5% afuco) Formulation 16 76.9 17.83.0 (incl. 5% afuco)

TABLE 10b Triantennary Triantennary + Name Biantennary 1 → 6 1 repeat tovalue 74.4 18.1 3.7 Formulation 10 71.4 19.3 4.0 Formulation 11 73.018.7 3.3 Formulation 12 72.3 19.7 3.4 Formulation 13 72.4 19.2 3.4Formulation 14 74.2 18.7 3.2 Formulation 15 73.0 18.7 2.8 Formulation 1674.3 19.7 3.2 (incl. 4% afuco)

TABLE 11a Name Degree of sialylation to value 88.3 Formulation 11 87.0Formulation 12 88.2 Formulation 13 85.8 Formulation 14 85.8 Formulation15 86.6 Formulation 16 86.9

TABLE 11b Name Degree of sialylation to value 88.3 Formulation 10 87.5Formulation 11 86.6 Formulation 12 87.7 Formulation 13 84.1 Formulation14 84.3 Formulation 15 85.7 Formulation 16 86.5

1. Liquid formulation consisting of human interferon-β as activeingredient, a buffer for setting a pH of 5 to 8, and one or more aminoacids, wherein the liquid formulation shows after storage for 3 monthsat 25° C. a long-term stability of the biological in vitro activity ofat least 80% of the initial activity, wherein the interferon-β comprisesat least one oligosaccharide structure with the proviso that theformulation does not comprise any acidic amino acids, arginine orglycine in amounts of between 0.3 and 5% by weight, and wherein theformulation contains no human serum albumin, surfactant or detergent. 2.Liquid formulation according to claim 1, wherein said buffer is forsetting a pH of 6 to 7.2.
 3. Liquid formulation according to claim 1,characterized in that the oligosaccharide structures are triantennal andmay comprise at least one N-acetyllactosamine repeat.
 4. Liquidformulation according to claim 1, characterized in that theoligosaccharide structure is triantennal and is linked at positions 1->4and/or 1>6.
 5. Liquid formulation according to claim 1, characterized inthat the proportion of oligosaccharide structures amounts to 0.5 to 3%,and wherein the oligosaccharide structures are tetraantennal.
 6. Liquidformulation according to claim 1, wherein the oligosaccharide structureis sialic acid and the sialic acid content is composed ofN-acetylneuraminic acid and N-glycolylneuraminic acid.
 7. Liquidformulation according to claim 6, characterized in that the sialic acidcomprises N-glycolylneuraminic acid and at least 90% N-acetylneuraminicacid.
 8. Liquid formulation according to claim 1, characterized in thatthe at least one oligosaccharide structure corresponds to one of thefollowing formulae:

in which NeuAc represents N-glycolylneuraminic acid.
 9. Liquidformulation according to claim 1, characterized in that it has aspecific activity of at least 200×10⁶ IU/mg.
 10. Pharmaceuticalpreparation containing a liquid formulation according to claim 1, in acompound formulated with a pharmaceutically acceptable vehicle orexcipient.
 11. Pharmaceutical preparation according to claim 10 in theform of unit doses of 3×10⁶ IU/ml to 10×10⁶ IU/ml.
 12. Pharmaceuticalformulation according to claim 10, characterized in that it is availablein forms appropriate for topical or parenteral administration.
 13. Thepharmaceutical preparation of claim 10, which has a shelf life that isgreater than a pharmaceutical preparation containing human interferon βand human serum albumin.
 14. The pharmaceutical preparation of claim 13,characterized in that the improved shelf life encompasses improvedlong-term stability of the biological in vitro activity, of the chemicalintegrity or/and of the physical integrity.
 15. Liquid formulationaccording to claim 1, wherein the liquid formulation contains IFN-β in aconcentration of 10×10⁶ IU/ml.
 16. A method for obtaining the liquidformulation of claim 1, comprising providing an aqueous solution ofhuman-IFN-beta in an impure state, wherein said aqueous solution doesnot contain human serum albumin, surfactant or detergent, subiectingsaid aqueous solution of human IFN-beta to liquid/liquid phaseextraction, then performing color affinity chromatography, performingmetal chelation chromatography, followed by size-exclusionchromatography, in which the chromatographic stages are carried out inthis order, then adding a buffer for setting a pH of 5 to 8 and addingone or more amino acids, with the proviso that the formulation does notcomprise any acidic amino acids, arginine or glycine in amounts ofbetween 0.3 and 5% by weight, thereby obtaining the liquid formulationof claim
 3. 17. The method of claim 16, characterized in that theliquid/liquid phase extraction is carried out with an aqueous two-phasesystem based on polyalkyleneglycol/dextran or polyalkyleneglycol/salt.18. The method of claim 17, characterized in that the polyalkyleneglycolused is polyethyleneglycol with a molecular weight of 1000 to 6000 orpolypropyleneglycol with a molecular weight of 1500 to
 4000. 19. Themethod of claim 17, characterized in that the salt used is NaCl, LiCl,NaI, KI, NA₂SO₄, NA₂HPO₄, K₂SO₄, NaH₂PO₄, KCl, NH₄Cl, (NH₄)₂SO₄, sodiumcitrate or sodium oxalate.
 20. The method of claim 17, characterized inthat the dyestuff used in affinity chromatography is Cibacron blue F 3GA.
 21. The method of claim 17, characterized in that the elution inaffinity chromatography is carried out using PBS containing 10 to 70% byweight of ethylene glycol and/or 20 to 50% by weight of propyleneglycol.
 22. The method of claim 17, characterized in that the elution inchelation chromatography is carried out using a competitive substance orNH4Cl or a pH gradient of around pH 7 to around pH 2 or by means ofisocratic elution with descending pH.
 23. The method according to claim22, wherein said competitive substance is selected from the groupconsisting of imidazole, histidine and glycine.
 24. The method of claim17, characterized in that the elution in size exclusion chromatographyis carried out with PBS containing 0 to 0.5 mol/l NaCl.
 25. The methodof claim 16, characterized in that the ions used in metal chelationchromatography are Cu²⁺, Zn²⁺, Co²⁺or Ni²⁺.
 26. The method of claim 16,characterized in that, for the size exclusion chromatography, separationmedia are used which have a separation range of 1 000 to 600 000daltons.
 27. The method according to claim 26, wherein said separationmedia are selected from the group consisting of Sephadex G150, SephadexG150 superfine, Sephacryl S-200 High Resolution, Superose 12 prep gradeand TSK-SW 3000.